SUMMARY: PROJECT 2 Transformation of castration resistant prostate cancer (CRPC) towards androgen signaling independence has emerged as a resistance mechanism in a subset of metastatic CRPC following exposure to androgen receptor (AR)-targeted therapies such as abiraterone or enzalutamide. Clinically, patients typically present with progression in the setting of a low or modestly rising serum prostate specific antigen (PSA) and metastatic biopsies can show pathologic or molecular features consistent with neuroendocrine prostate cancer (NEPC). NEPC is associated with low or absent AR expression, suppressed AR signaling, retention of early genomic mutations from its adenocarcinoma precursor, and acquisition of distinct genomic and epigenomic alterations (Beltran et al., Nature Medicine, in press). The development of novel therapeutic approaches for patients with NEPC represents a clinical unmet need. Over the last six years, our group has focused on characterizing the molecular landscape of NEPC and have identified and validated new therapeutic targets, including the N- Myc/Aurora A pathway and specific epigenetic modifiers such as (Enhancer of Zeste Homolog 2) EZH2. Our overarching hypothesis is that N-Myc cooperates with both Aurora-A and EZH2 to drive the neuroendocrine phenotype and that characterizing this driving role will lead to more effective targeting strategies for this tumor entity. To address this hypothesis we propose to characterize the interaction between the EZH2 and N-Myc signaling in driving NEPC and will also build on prior work evaluating allosteric inhibitors of the Aurora-N-Myc complex (e.g., MLN8237) and the EZH2 inhibitors to develop more effective combination strategies to target NEPC (Aim 1). In addition, we aim to develop novel allosteric compounds targeting N-Myc/Aurora-A complex through our collaboration with the Tri-Institutional Therapeutics Discovery Institute at WCM (Aim 2). Finally we will develop clinical biomarkers to predict response in targeting N-Myc and EZH2 in CRPC. We will evaluate pre-treatment metastatic biopsies from patients with NEPC enrolled in a Phase 2 study of the aurora kinase A inhibitor MLN8237 (an allosteric inhibitor of the Aurora-N-Myc complex) and a Phase 1 trial of the EZH2 inhibitor GSK146 and correlate AR and N-Myc signaling determined by RNA-seq, Aurora-N-myc-EZH2 complex formation, and EZH2 target gene expression with clinical response (Aim 3). Our goal is to develop more effective targeting strategies for a biomarker-selected subgroup of late stage CRPC driven by N-Myc and less dependent on the AR. At the end of this project we will have a better understanding of the mechanisms underlying N-Myc/EZH2 driven NEPC and we will have identified biomarkers of response to N-Myc and EZH2 inhibition. This study will serve as a solid preclinical foundation for the development of new biomarker-driven therapeutic strategies for treating patients with advanced prostate cancer.